Plant Physiol. (1999) 120: 623-632
Gibberellin Dose-Response Curves and the Characterization of
Dwarf Mutants of Barley
Peter M. Chandler* and
Masumi Robertson
Commonwealth Scientific and Industrial Research Organization Plant
Industry, G.P.O. Box 1600, Canberra, ACT 2601, Australia
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ABSTRACT |
Dose-response curves relating gibberellin (GA) concentration
to the maximal leaf-elongation rate (LERmax) defined three
classes of recessive dwarf mutants in the barley (Hordeum
vulgare L.) `Himalaya.' The first class responded to low
(10
8-106 M)
[GA3] (as did the wild type). These grd
(GA-responsive dwarf) mutants are
likely to be GA-biosynthesis mutants. The second class of mutant,
gse (GA sensitivity), differed
principally in GA sensitivity, requiring approximately 100-fold higher
[GA3] for both leaf elongation and 
-amylase production
by aleurone. This novel class may have impaired recognition between the
components that are involved in GA signaling. The third class of mutant
showed no effect of GA3 on the LERmax. When
further dwarfed by treatment with a GA-biosynthesis inhibitor, mutants
in this class did respond to GA3, although the
LERmax never exceeded that of the untreated dwarf. These
mutants, called elo (elongation), appeared
to be defective in the specific processes that are required for
elongation rather than in GA signaling. When sln1
(slender1) was introduced into
these different genetic backgrounds, sln was epistatic
to grd and gse but hypostatic to elo. Because the rapid leaf elongation typical of
sln was observed in the grd and
gse backgrounds, we inferred that rapid leaf elongation is the default state and suggest that GA action is mediated through the
activity of the product of the Sln gene.
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INTRODUCTION |
Dwarf mutants have proven to be valuable tools in
hormone studies in a wide range of plant species. This has been
particularly so for the GAs (for review, see Ross et al., 1997
) and
more recently for the brassinosteroids (Clouse and Sasse, 1998). The
identification of hormone-biosynthetic mutants that are normalized by
hormone application illustrates the importance of such hormones in
determining plant stature. Therefore, when characterizing new dwarf
mutants, the growth response after hormone application is an important first screen because it allows potential hormone biosynthetic mutants
to be identified. However, a considerable proportion of new mutants may
show either no or only partial growth response to applied hormone; and
these are potentially altered in signal transduction or in processes
affecting growth. For the GAs, several such classes of dwarf can be
recognized (Ross et al., 1997). In addition, there are GA-signaling
mutants that show either a constitutive or an enhanced GA response
(Ross et al., 1997).
Current interpretations of this broad grouping of GA "response"
mutants are imprecise, largely because plant growth is regulated by
many factors in addition to GA. Therefore, failure to respond to
applied GA does not necessarily mean a deficiency in GA signaling. For
example, two pea mutants that were originally interpreted as
GA-response mutants were later shown to be brassinosteroid deficient
and nonresponsive (Nomura et al., 1997). It is clearly an advantage to
use, when possible, an independent GA response that does not involve
growth; in this respect cereal systems have proved valuable because
they allow changes in leaf growth to be compared with -amylase
production by aleurone tissue (Gale and Marshall, 1973; Chandler, 1988;
Lanahan and Ho, 1988).
Another problem arises because the final extent of a response may not
be the most accurate measure of hormone responsiveness. Nissen (1988)
analyzed data in the literature for several GA responses, including
leaf elongation, and concluded that they were "almost uniformly
subsensitive"; i.e. a greater-than-expected concentration range of
applied GA was required for the response to go from 10% to 90% of
maximal values. Weyers et al. (1987, 1995) have emphasized the
importance of determining the initial or maximal rates of response to
hormone application rather than the final extent, but despite the
renewed interest in hormone-response mutants (largely because of
studies using Arabidopsis), this approach has not been widely adopted.
When a reduced rate of response to hormone application is observed, it
is necessary to determine whether there has also been a change in the
concentration range over which the response occurs. In this manner, a
mutant that requires higher hormone concentrations than the wild type
does to bring about a similar response can be distinguished from one
with a reduced response capacity (Firn, 1986).
Hormone dose-response curves therefore provide data essential for
characterizing GA-response mutants (Weyers et al., 1995; Swain and
Olszewski, 1996). Where the responses being measured are complex (such
as organ-elongation rates) and likely to integrate a number of
"simpler" components, it is important that mutants be compared with
the appropriate wild-type background. In the case of an induced
mutation, backcrossing is required so that the possibility is reduced
that independent mutational events contributed to the response being
measured. In this study we describe a leaf-elongation assay for GA
responsiveness that defines three classes of dwarf mutants in barley:
grd (GA-responsive
dwarf), gse (GA
sensitivity), and elo (elongation).
Genetic interactions of these mutants with the sln
(slender) "constitutive GA response" mutant
(Foster, 1977) suggest that GA signaling proceeds through the SLN gene
product.
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MATERIALS AND METHODS |
Chemicals
Initial experiments used commercial preparations of
GA3 (>90%, Sigma), but to obtain saturation in
dose-response experiments, very high (millimolar) concentrations were
required. Considerable GA1 was detected by
GC-selected ion monitoring in the commercial GA3 preparation, so pure preparations of
GA1 and GA3 were kindly provided by L.N. Mander, Research School of Chemistry,
Australian National University, Canberra, ACT. Samples of
GA44, GA19, and GA20 were also kindly provided by L.N. Mander.
All GA solutions were prepared by dissolving powder in 1 mM
potassium-phosphate buffer, pH 5.5, and diluting in this solution when
necessary. The stock solution of GA3 was 9.76 mM, and excess acidity due to GA3 was
neutralized by the dropwise addition of 20 mM KOH until the
pH returned to 5.5. GA solutions were stored at 
20°C. Tetcyclacis (94.8%) was kindly provided by Dr W. Rademacher (BASF, Limburgerhof, Germany) and dissolved in ethanol at 3.5 mM.
Plant Material
The dwarf mutants of barley (Hordeum vulgare)
`Himalaya' were isolated after mutagenesis with sodium azide as
described by Zwar and Chandler (1995). From about 200 independent dwarf
mutants we selected several different types for detailed study, based on GA3 application and genetic-complementation
studies. Seven mutants are described here, all of which are recessive
and all of which have been through three back-crossing generations
before the establishment of homozygous seed stocks; at the seedling
stage they ranged in height from approximately 20% to 50% of the
wild-type parent. The first class (M117, M359, and M411) showed a large response to GA3 application (microdrops or
spray), and the three mutants represent three genetic loci.
Phenotypically these mutants are similar, with leaves that are shorter
and darker green and stem internodes that are shorter than the wild
type. The second class (M121 and M488) showed only partial growth
responses to GA3, even at high concentrations.
These two mutants are phenotypically similar to those described above,
and represent two alleles at a single locus. The third class (M21 and
M626) did not show any growth response to GA3,
and the two mutants are at different genetic loci.
The introduction of the sln mutation into the wild type and
M117 was described by Smith et al. (1996). The same procedure was
followed in crossing sln1 into the other classes described here. In one class (M121 and M488) the sln phenotype is
expressed as it is in the wild type and in M117. In the other class
(M21 and M626) the sln phenotype is not observed during
growth of the first two leaves, but homozygous (sln1sln1)
plants could be identified later in their growth on the basis of
abnormal stem elongation. Plants homozygous for sln1 do not
set seed, so comparisons were made between phenotypically normal
(Sln1) and slender (sln1sln1) segregants in the
progeny of Sln1sln1 heterozygotes in different genetic
backgrounds.
Seedling Growth and Determination of LERmax and
Final Blade Length
Grains were surface-sterilized as described previously (Chandler
and Jacobsen, 1991) and placed embryo-side down between two sheets of
autoclaved paper (3MM, Whatman) "envelopes" that were moistened
with the appropriate solution and held vertically in a plastic frame
placed in the solution. After the grains were stratified (4°C in the
dark) for 48 h, they were placed under low-intensity fluorescent
lighting at 20°C (d 0). After a further 3 d, the germinated
grains in each envelope were culled for uniformity of shoot length
(providing approximately 15 seedlings per sample), and the envelope was
aligned with positional markers on a clear plastic sheet. The position
of the tip of each leaf was marked on the plastic sheet, and the
envelope was returned to the growth assembly. After an additional 1, 2, 3, and 4 d, the envelopes were again placed on the original sheets
in their original position, and the new position of each leaf tip was
marked. For each 24-h interval, the distance between marks was recorded
and the mean length increment was determined. In most cases the maximal
elongation occurred between d 4 and 5 after transfer from 4°C to
20°C. Occasionally, maximal growth occurred in either the previous or
the following 24-h interval. For each genotype and treatment, the
maximal value in each set of daily increments was expressed as a
millimeter-per-day rate and abbreviated LERmax.
In one experiment, seedlings of M117 and M411 were maintained until the
growth of L1 had ceased. The shoot was then
dissected to determine the final blade length.
Construction of Dose-Response Curves
LERmax data were analyzed as a function of
[GA] using individual seedling data and PEST software (Weyers
et al., 1987). This program provides estimates of hormone-sensitivity
parameters fitted to a modified Hill equation. The curves fitted to the
data by the PEST software were plotted together with raw data points
representing the means ± SE of
LERmax.
-Amylase Determinations
The embryonic axes of dry grains of the wild type and M488 were
removed using a dissecting blade under a dissecting microscope. There
was minimal damage to the scutellum. "De-axised" grains do not
produce -amylase unless incubated with GA3
(Jones and Armstrong, 1971), and any -amylase produced remains
within the grain. The de-axised grains were surface-sterilized, placed
in paper envelopes, and incubated under conditions identical to those for intact grains (see above). -Amylase activity was determined as
previously described (Chandler and Jacobsen, 1991) on duplicate samples
of five grains each.
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RESULTS |
Growth of L1
Elongation of L1 initially involved only the
blade, but between d 5 and 7 both blade and sheath were elongating
(Fig. 1). At later stages, elongation of
the leaf involved only the sheath. The rate of leaf elongation was far
from uniform, and LERmax occurred just before
blade growth began to slow (just before sheath growth commenced; Fig.
1, inset). Maximal rates of sheath elongation did not exceed those of
the blade (data not shown).
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| Figure 1.
Growth of L1 of wild-type barley.
Grains were surface-sterilized, placed in moist paper envelopes,
stratified, and incubated as described in ``Materials and Methods''.
At the indicated times the mean L1 lengths of 10 seedlings
were determined, as well as the mean lengths of the blade and sheath.
Where not visible, error (SE) bars lie within the symbols.
Inset, Mean elongation rate of the blade of L1 in the
previous 24 h was plotted as a function of days of growth. The
LERmax value for this set of data is indicated.
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L1 of the dwarf mutants was always smaller than
that of the wild type, and LERmax values were
considerably lower (7-15 mm d1, compared with
37 mm d1 for the wild type; see below).
However, the pattern of L1 growth of the mutants
was similar to that of the wild type; blade elongation preceded that of
the sheath, and LERmax occurred just before the transition from blade growth to sheath growth (data not shown).
Effects of GA3 on LERmax
Grains of the wild type and of the dwarf mutants were germinated
in a range of concentrations of GA3, and the
LERmax was determined. The resulting
dose-response curves (Fig. 2) show an
LERmax for the wild type of about 37 mm
d1 at low [GA3]
(<108 M), increasing to about 67 mm d1 at high [GA3]
(>106 M). Among the dwarf
mutants, three response classes were identified that differed in
the effect of GA3 on
LERmax: the first class (M117, M359, and M411)
responded to GA3 over the same concentration range (108-106
M) as the wild type; the second class (M121 and M488)
responded over a much higher and wider concentration range
(106-103
M); and the final class (M21 and M626) showed no response
to [GA3] as high as 103
M. Growth rates on the lowest concentrations of
GA3 were not significantly different from rates
on control medium without GA3 (compare Figs. 2,
6, and 7).
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| Figure 2.
Dose-response curves relating LERmax
of L1 to [GA3]. Grains of the indicated lines
were surface-sterilized, placed in paper envelopes moistened with the
appropriate [GA3], stratified, incubated in low light,
and LERmax (mean ± SE) of seedling
L1 was determined as described in ``Materials and Methods''. Curves in the top six panels were fitted using PEST
software (Weyers et al., 1987). Note differences in the range of
GA3 concentrations in different panels.
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| Figure 6.
LERmax of L1 of
elo mutants and a grd mutant growing with
or without tetcyclacis and GA3. Grains of M21
(elo1), M626 (elo2), and M117
(grd1) were surface-sterilized, placed in moist paper
envelopes containing, where appropriate, 2 µM tetcyclacis
or 2 µM tetcyclacis plus 10 µM
GA3, stratified, and incubated under low light; and the
mean LERmax of seedling L1 was determined as
described in ``Materials and Methods''. A replicate consisted of 10 seedlings, and there were three replicates for each genotype and
treatment. Within a genotype, letters (a, b, or c) indicate
significance (P < 0.05) for the differences between the means of
each treatment.
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| Figure 7.
LERmax of L1 of
Sln1 and sln1sln1 segregants in
different genetic backgrounds. Grains from stocks in which the
sln1 allele was segregating in different genetic
backgrounds (WT, wild type; grd1, M117;
gse1, M121; and elo1; M21) were
surface-sterilized, placed in moist paper envelopes, stratified, and
incubated in low light, and the LERmax (means ± SE) of seedling L1 was determined as described
in ``Materials and Methods''. In an elo1 background,
slender (sln1sln1) seedlings cannot be distinguished at
the first leaf stage from Sln1 seedlings, but after
transplanting and further growth, the early stem elongation
characteristic of sln1sln1 plants, which still occurs in an
elo1 genetic background, allowed the genotype to be
determined.
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For the wild type and the first two classes, the mean values of
different parameters were estimated from the fitted curves: LERmax at zero and saturating concentrations of
GA3 (Rmin and Rmax, respectively); the Hill interaction
coefficient (p), which provids a measure of the
"steepness" of the response to increasing concentration of
GA3; and [H]50, the
concentration of GA3 at which 50% of the maximal
response to GA3 is attained (Table
I). The values of these parameters
highlight the similarity between the wild type and the first class of
mutant in their response to GA3, with
p values close to unity (reflecting near Michaelis-Menten behavior), and the estimated concentration of GA3
required for a half-maximal response falling within a narrow range of
56 to 120 nM. The RMIN
values of the three dwarf mutants in this class were lower than those
of the wild type, which is consistent with their dwarf nature, but
their Rmax values were not quite as high as those
of the wild type, possibly because the grains of the dwarf mutants were
smaller by 5% to 20% on a dry-weight basis (data not shown). As a
consequence, in mature grains the L1
primordium of the dwarf would probably be smaller than that
of the wild type, and its capacity to increase the elongation rate in
response to exogenous GA could potentially be compromised.
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Table I.
Parameters estimated from GA3
dose-response curves
Mean values (and their 95% confidence limits) of parameters (see text
for explanation of symbols) were estimated for the fitted curves shown
in Figure 2 using PEST software (Weyers et al., 1987).
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The values shown in Table I contrast the behavior of the wild type and
the first class of mutants (M117, M359, and M411) against that of the
second class (M121 and M488). In particular, there was a much broader
response in the latter mutants (p values considerably less
than unity), and much higher (90- to 350-fold) concentrations of
GA3 were required for half-maximal response ([H]50). Rmax values for
these mutants were again somewhat less than those for the wild type,
and an argument similar to that above could be made based on grains
that are approximately 25% smaller (dry weight) than those of the wild
type. In addition, even at 102
M GA3 (the highest
concentration tested), the response may not have been saturated. The
estimated values of the parameters in Table I for this second class of
mutant might be less reliable than those estimated for the wild type
and the first mutant class because saturation was barely attained.
Nevertheless, the main differences (a broader transition and a
displacement of the response to higher [GA3])
are clearly discernible in the curves of Figure 2.
Similar experiments were carried out with GA1,
which is also an important bioactive GA for leaf elongation. A less
extensive range of concentrations and mutants was studied, but the
results (data not shown) were in close agreement with those described for GA3. The principal difference was that the
wild type and the first class of mutant had an
[H]50 value for GA1
(500-1700 nM) that was approximately 10-fold higher than
that estimated for GA3. If we assume that
GA1 and GA3 have equal
intrinsic activity and similar rates of uptake, the difference in
[H]50 values may reflect more rapid catabolism
of GA1. The behavior of the three mutant classes
in their responses to GA1, including the
estimates for p and the relative differences in
[H]50, paralleled the behavior observed for
GA3.
Comparing the Effects of GA3 on LERmax and
on Final Blade Length
Seedlings of two mutants (M117 and M411) were maintained on a
range of GA3 concentrations until growth of
L1 stopped, allowing the final length of the
L1 blade to be determined. To allow a direct
comparison between the effects of GA3 on
LERmax and on final blade length, each response
was normalized to an RMIN value of 1 (Fig.
3). Final blade length responded to
increasing concentration of GA3, but the response
was lower in magnitude than that of LERmax and
occurred over a wider range of GA3
concentrations. These effects may have resulted from differences in the
duration of elongation.
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| Figure 3.
Dose-response curves comparing the effects of
GA3 on either LERmax or final blade length.
Grains of the indicated lines were surface-sterilized, placed in paper
envelopes moistened with the appropriate [GA3],
stratified, and incubated in low light, and LERmax
(mean ± SE) or final blade length of seedling
L1 was determined as described in ``Materials and Methods''. To allow direct comparison, response ratios are plotted in
which the LERmax or L1 blade length in the
absence of GA3 is assigned a value of unity. Curves are the
same (minus data points) as shown in Figure 2. Individual data points
( ) are for final blade length.
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Definition of Mutant Classes
Based on the dose-response curves (Fig. 2), three classes of dwarf
mutants can be defined. The first, grd, responds to
GA3 over the same concentration range as the wild
type. These mutants are proposed to have normal GA signaling and their
dwarfism is associated with low levels of endogenous bioactive GAs
(P.M. Chandler and J.R. Lenton, unpublished results). The three mutants
in this class represent three different genetic loci: grd1
(M117), grd2 (M359), and grd3 (M411). Mutants in
the second class are primarily characterized by an alteration in GA
sensitivity (the gse mutants). M121 and M488 represent
alleles at the gse1 locus, because no complementation is
observed when they are intercrossed. The greatly reduced sensitivity to
GA of these two mutants probably explains why they showed only poor
growth responses to GA3 in preliminary experiments (see ``Materials and Methods''). Mutants in the third
class show no elongation response to GA. On the basis of the results
presented below, these mutants are proposed to be defective in the
specific processes that are required for leaf elongation (the
elo mutants), rather than in GA signaling. M21 and M626
represent different elo loci. For each of these three mutant
classes, additional experiments aimed at a more detailed
characterization were performed.
Response of grd Mutants to GA-Biosynthetic
Intermediates
GA-biosynthetic intermediates may be active or inactive in
promoting elongation in dwarf mutants, depending on the concentration at which they are applied, the severity of the dwarfing mutation, and
the step in the GA biosynthetic pathway in which the mutant is blocked.
The growth responses of L1 of the three
grd mutants to late intermediates of the early
13-hydroxylation pathway (Grosselindemann et al., 1992) were determined
(Fig. 4). For each mutant,
GA1 treatment resulted in
LERmax values that were greater than the
wild-type values (approximately 37 mm d1). The
[GA] used in these experiments (2 × 106
M) was only slightly greater than the
[H]50 value determined for
GA1 (approximately 1 × 106 M) so that
LERmax would be highly responsive to the content
of active GAs. GA20 was very effective in
stimulating LERmax of the grd1 and
grd3 mutants, and GA44 and
GA19 were slightly less effective. This pattern
differed markedly from that seen for the grd2 mutant, in
which each of the intermediates had very low activity in stimulating elongation. We inferred from this that the grd1 and
grd3 mutants convert "inactive" GA precursors such as
GA20 to growth-active GAs, whereas
grd2 mutants do not (or do so at a greatly reduced rate).
This pattern would be consistent with grd2 mutants having reduced levels of 3
-hydroxylation (converting
GA20 to GA1), whereas the
other two loci are presumably blocked earlier in the pathway.
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| Figure 4.
LERmax (means ± SE)
of different grd mutants in the presence of
GA-biosynthetic intermediates. Grains of M117 (grd1),
M359 (grd2), and M411 (grd3) were
surface-sterilized, placed in paper envelopes moistened with the
indicated GA at 2 µM, stratified, and incubated in low
light, and LERmax (means ± SE) of
seedling L1 was determined as described in ``Materials and Methods''. GA44, GA19, and
GA20 are successive biosynthetic intermediates in the early
13-hydroxylation pathway leading to the formation of the bioactive
GA1.
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-Amylase Production by gse1 Grains in Response to
GA3
Mutants in the gse1 locus were characterized by reduced
sensitivity to GA3 for leaf elongation;
therefore, -amylase production by aleurone tissue was also examined.
-Amylase activity in de-axised wild-type grains increased with time
in the presence of 108 to
107 M
GA3 at approximately one-half the maximal rate
observed with 106 and
105 M
GA3 (Fig. 5). In
contrast, -amylase activity of de-axised gse1 grains
showed no increase with time at GA3
concentrations 
106
M, intermediate rates of accumulation with
105 M
GA3, and high rates of accumulation at
104 to 103
M GA3. This pattern
parallels that observed for leaf elongation, in which responses
equivalent to those of the wild type required at least 100-fold-higher
concentrations of GA3. The maximal rate of
-amylase accumulation in the mutant was less than that of the wild
type, possibly because the grains were 25% smaller (on a dry-weight
basis) than those of the wild type, and perhaps there was an equivalent
reduction in aleurone cell number. We concluded that the
gse1 mutants are defective in a component of GA signaling that is required for two independent GA responses: leaf elongation and
-amylase production by aleurone.
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| Figure 5.
-Amylase activity of de-axised grains of the
wild type and a gse1 mutant incubated with different
[GA3]. The embryonic axes of wild type and M488
(gse1) grains were removed, and the resulting de-axised
grains were surface-sterilized, placed in paper envelopes moistened
with the indicated [GA3], stratified, and incubated in
low light as described in ``Materials and Methods''. At the indicated
times, duplicate samples (five grains each) were harvested and
-amylase was extracted and assayed. Each data point is the mean of
duplicate samples.
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Conditional Regulation of LERmax in elo
Mutants by GA3
The two elo mutants were characterized by low rates of
leaf elongation even at very high concentrations of
GA3 (Fig. 2); however, the aleurone of both
mutants showed near-normal responses to GA3 for
-amylase production (data not shown). We considered the possibility that leaf-elongation rates were limited by defective components involved in leaf elongation rather than in GA signaling. Inhibitors of
GA biosynthesis such as tetcyclacis induce dwarfing in barley, but this
effect can be overcome by GA3 (Zwar and Chandler,
1995). Grains were germinated of the two elo mutants and of
the grd1 mutant (as a control), and the seedlings were grown
in control conditions in the presence of tetcyclacis alone or
tetcyclacis plus GA3.
Significant additional dwarfing was induced in all of the lines by
tetcyclacis treatment, as revealed by the LERmax
values (Fig. 6). When
GA3 was also present, the
LERmax values returned to control levels for both
elo mutants but greatly exceeded control levels for the
grd1 mutant (as expected). We inferred from this that the
elo mutants are capable of responding to
GA3 provided leaf elongation occurred at a lower
rate than in control conditions. In more detailed experiments with the
elo1 mutant, the concentration dependence for restoration of
LERmax by GA3 was examined.
The results (data not shown) indicated that in the presence of
tetcyclacis, concentrations of GA3 as low as
107 M were able to
restore LERmax to control levels, indicating that the elo mutants were capable of responding to low
concentrations of GA3. The failure of such
mutants to respond to GA3 in the dose-response experiment (Fig. 2) was presumably because they were already elongating at their maximal rate.
Leaf Elongation of the sln1 Mutant in Different
Genetic Backgrounds
The sln1 mutant of barley (Foster, 1977) exhibits rapid
leaf elongation without added GA3, yet has lower
than normal levels of active GAs in its leaves (Croker et al., 1990).
On this basis, and because it shows high rates of -amylase
production by aleurone incubated without added
GA3, sln1 is regarded as a
constitutive GA-response mutant (Chandler, 1988; Lanahan and Ho, 1988).
We previously showed that sln1 derivatives of M117
(grd1) elongated rapidly despite the dwarfing background
(Smith et al., 1996). Similar results were found for sln1 in
grd2 or grd3 backgrounds (P.M. Chandler,
unpublished data). To determine whether double mutants of
sln1 with either gse1 or elo would
also elongate rapidly without added GA3,
LERmax values were determined for segregating Sln1 and sln1sln1 types in the different
genetic backgrounds. The results (Fig. 7)
show that sln1 homozygotes elongate equally rapidly in the
wild type, grd1, and gse1 genetic backgrounds, indicating that sln1 is epistatic to gse1 and
grd1. In contrast, the LERmax value of
sln1 homozygotes in an elo1 (Fig. 7) or
elo2 genetic background (data not shown) did not differ
significantly from the Sln1 segregants, indicating that
sln1 was hypostatic to these two elo loci, which
is consistent with the proposal that these mutants were already
elongating at their maximal rate. The effect of homozygosity at
sln1 on LERmax values for the wild
type, grd1, and gse1 was equivalent to that of
saturating concentrations of GA3 (compare Figs. 2
and 7).
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DISCUSSION |
Hormone dose-response studies on the effects of differing
concentrations of GA on LERmax provided an
effective basis for discriminating between different classes of dwarf
mutants in barley. In earlier studies GA application to seedlings had
readily distinguished fully responsive dwarfs (thought to be affected
in GA biosynthesis) from mutants that showed no response to GA. Dwarf
mutants that gave a partial response to GA were problematic, because
they may have involved alterations either in the magnitude of response at saturating hormone concentrations or in the concentration range over
which a response occurred. Dose-response experiments distinguished between these possibilities. The gse mutants fit the latter
category, and thereby define a novel type of mutant that is involved in GA signaling. Growth responses to GA in a recently described pea mutant
(lgr) have similar properties (Ross et al., 1997).
The current interest in hormone signal transduction requires that
quantitative assays be used to characterize the mutants that are
affected in such processes. The GA dose-response curves described here
provide a framework for future characterization of the remaining barley
dwarf mutants in our collection. Sensitivity parameters were estimated
from the dose-response curves using PEST software (A'Brook, 1987;
Weyers et al., 1987), which fits data to a modified Hill equation. It
was significant that near Michaelis-Menten responses were observed even
for growth rates of whole leaf blades (at least for the wild type and
grd mutants), because there are presumably many steps
between GA perception and the final leaf growth rate at which the
initial magnitude of a response to GA could be modified.
In many previous studies there was a broader-than-expected GA
concentration range over which responses occurred (Nissen, 1988). In
contrast, the range of GA concentrations over which a response occurred
in the wild type and the grd mutants was relatively narrow (p 
1; see Table I). This difference was probably
because we used LERmax as a measure of hormone
response (Weyers et al., 1987) rather than the extent of response,
which was used in the earlier studies. In some cases such broad
transitions may be genuine, perhaps reflecting attenuation so that a
wide range in hormone content can be accommodated. However, until they
are analyzed in terms of rate rather than final extent, the wide
concentration range might also be misleading. When we monitored final
blade length rather than LERmax, a broader
transition was observed and the magnitude of the response was smaller
(Fig. 3). The probable explanation for this difference is a shorter
duration of the response at high concentrations of
GA3, so that the effect of
GA3 on growth rate was never exactly matched by
the effect on final length.
A [GA3] of approximately
107 M stimulated
LERmax in grd dwarfs to that of the
wild type given only water. This concentration is close to the
[H]50 value estimated for
GA3 (Table I), a condition in which
LERmax changes most rapidly as the concentration
of applied GA3 changes. In this range there was a
20% change in LERmax for a 2-fold change in
[GA3], illustrating the potential for
relatively small changes in the content of endogenous bioactive GAs to
have considerable effects on the leaf-elongation rate when, for
example, plants respond to different environmental factors. It is
difficult to compare the [GA3] applied in a
treatment (e.g. 107 M)
with the endogenous contents of bioactive GAs, because we know neither
the relative contributions of different GAs (GA1, GA3, and possibly other GAs) in determining leaf
growth rate, nor the most appropriate part of the leaf (zones of cell
division or elongation, or perhaps only the epidermis of such regions) in which to determine GA content.
Tonkinson et al. (1997) determined GA1 and
GA3 contents in the elongation zones of the
second leaves of wheat seedlings and, assuming uniform distribution,
their values correspond to 2 to 7 nM. These estimates are
considerably lower than the [H]50 values above,
but there are many factors that could account for such a discrepancy,
including species and leaf differences and the assumption of uniform GA
distribution. It is apparent that the maintenance of normal growth
rates requires an adequate supply of and an ability to sense endogenous
GAs, because mutants that affect either process are dwarfed. The
relative importance of these two processes in explaining natural
variation in growth rate is difficult to assess. Weyers et al. (1995),
in discussing hormonal control in a general sense, argued that combined
control should always be assumed unless there is evidence to the
contrary. In this context it is interesting that the growth rate of the grd1gse1 double mutants was considerably lower than either
of the single mutants (P.M. Chandler, unpublished observations).
Interpretation of the grd mutants is relatively
straightforward, because equivalent mutants have been isolated in a
range of other plant species, and have generally involved mutations in
the GA-biosynthetic pathway (for review, see Ross et al., 1997). For
example, the growth responses of the grd2 mutant (Fig. 4) are typical of 3-hydroxylase mutants that have been isolated in
several species. There are two GA-responsive dwarf mutants in barley
that have been studied in some detail (Hentrich et al., 1985; Boother
et al., 1991), and both of these are allelic with the grd1
locus described here (P.M. Chandler, unpublished data).
An important advantage of barley and some other cereals is the
availability at the seedling stage of two well-defined GA responses (leaf elongation and -amylase production) that involve different components (the meristem-leaf-elongation zone and aleurone,
respectively). This has been important in interpreting the
gse mutant category. In both assays the gse
mutants were capable of responding to GA3, probably to the same extent as the wild type and grd
mutants, but the gse mutants required approximately 100-fold
higher concentrations of GA3. These recessive
mutants are unique in showing reduced sensitivity for two different GA
responses. One interpretation is that they define receptors that have a
lower affinity for GA than the receptors in the wild type. Loeb
and Strickland (1987) showed that dose-response curves can reflect the
activity of components involved in signal transduction, rather than
initial receptor-hormone interactions; thus the gse1 mutants
may also involve changes in the downstream components of GA signaling.
An alternative interpretation is that a "primary" GA receptor or
signaling pathway is rendered nonfunctional in the gse
mutants, and the activity of a redundant pathway(s) with different
properties is revealed. An interesting feature of the dose-response
curves of both gse mutants was the broader range of
concentrations over which the response occurred (Fig. 2). The
associated lower values of p (Table I) may result from
negative cooperativity in the binding of interacting components (e.g. a
ligand to its receptor), either as a result of mutational change or
because a different signaling pathway was operating.
Two other interpretations of the gse phenotype are possible.
The first, involving overproduction of an enzyme that inactivates GAs,
is considered unlikely for several reasons: (a) we would expect the
trait to show some degree of dominance if it resulted from increased
levels of a catabolic enzyme; (b) the dose-response curves indicate
that extremely high concentrations of GA3 (by in
vivo standards) are still subsaturating, yet during normal growth the
gse mutants are not severely dwarfed, and an altered enzyme
that was capable of inactivating such high concentrations of exogenous
GA3 might be expected to have an extreme effect
on the endogenous GA content, resulting in a much more severe dwarf phenotype; and (c) determination of the endogenous GA content of
developing grains of M488 exhibits a profile that is very similar to
that of the wild type (P.M. Chandler, unpublished data), indicating that there are no major changes in GA metabolism. This includes stages
when the gse phenotype of developing grains is being
expressed, revealed by the failure of 10 µM
GA3 to induce germination of isolated immature
grains.
The second alternative interpretation of the gse mutants is
that their reduced sensitivity to GA may have resulted either from
increased levels of endogenous ABA or from enhanced responses to ABA
(Cutler et al., 1996), because in barley grains and seedlings, ABA
antagonizes many of the effects of GA. This interpretation is difficult
to exclude until more information is available, but two lines of
investigation have failed to provide support: first, the quantitative
hormone analysis of developing grains of M488 (see above) revealed ABA
contents similar to those of the wild type, and second, there were
similar relative reductions in the L1 growth rate
observed when gse1, grd1, and wild-type grains were germinated in the presence of 1 µM ABA
(P.M. Chandler, unpublished data).
The two elo mutants showed no significant growth stimulation
by GA3, yet their ability to perceive and
initially respond to GA3 was probably not
affected. For example, LERmax was responsive to
GA3 with approximately the normal concentration
dependence when the mutants were further dwarfed either by chemical
means (Fig. 6) or by making double mutants with a grd locus
(P.M. Chandler, unpublished data). This observation and the epistasis
of elo to sln1 (Fig. 7) suggest that the
mutations affect specific components required for leaf elongation
rather than those involved in GA signaling. There was no restoration of
normal growth when these mutants were germinated on other
growth-related hormones such as brassinolide, IAA, or kinetin (P.M.
Chandler, unpublished observations).
The characterization of these dwarf mutants suggests that they are
representative of three broad areas involving GA control of growth: GA
biosynthesis (grd), GA signaling (gse), and the growth processes themselves (elo). In the simplest model, GA
elicits a positive signaling pathway and growth is stimulated.
According to this model, the low growth rates of the grd and
gse mutants are due to the effects of reduced GA content and
GA sensitivity, respectively, on GA signaling. The sln1
mutant is recessive, and slender plants show "constitutive" GA
responses. Thus, the product of the wild-type Sln1 gene
(SLN) presumably functions as a negative regulator of GA signaling (if
sln1 involves a loss of function). Is SLN a negative
regulator that plays a direct role in GA signaling, or does it play an
indirect role? Other signaling pathways in the plant could modulate
flux through a positive GA-signaling pathway via SLN acting as a
negative regulator of this pathway.
Slender derivatives of the grd mutant showed a typical
slender phenotype rather than a dwarf phenotype. The same result was observed with slender derivatives of the gse mutant. Thus,
in an sln1 background, mutations such as grd and
gse that result in reduced GA signaling had no effect on
growth rate. If SLN is an indirect negative regulator of a positive
GA-signaling pathway, we might still expect to see reduced growth rates
in the double mutants because of reduced GA signaling. We favor the
view that SLN is a negative regulator whose activity is directly
involved in GA signaling. If the GA-signaling pathway is under negative control, the positive responses observed when, for example, GA is
applied must involve reducing the extent of negative regulation mediated by SLN. In the same manner, `Himalaya' barley grows at wild-type rates because with wild-type levels of GA signaling, it can
substantially reduce the extent of negative regulation imposed by SLN.
By contrast, the grd and gse mutants have lower levels of GA signaling and are less able to reduce the extent of
negative regulation by SLN, and consequently their growth is slow.
This interpretation is similar to that reached for the product of the
GAI gene in Arabidopsis, which, according to genetic evidence, also functions as a negative regulator involved in GA signaling and whose activity is proposed to be regulated by GA (Peng et
al., 1997; Harberd et al., 1998). In Arabidopsis there are now three
different proteins that, on the basis of mutant studies, are proposed
to be negative regulators of GA signaling. GAI (Peng et al., 1997) and
RGA (Silverstone et al., 1997) are closely related; based on sequence
comparisons, they are putative VHIID transcription factors. SPY is a
protein with a sequence closely related to O-linked GlcNAc
transferases (Jacobsen et al., 1996). Only for SPY has the proposed
role as a negative regulator of GA signaling been confirmed: transient
expression of barley SPY (HvSPY) largely prevented GA-induced
-amylase promoter activity in aleurone (Robertson et al., 1998). Is
barley SLN related to these other proteins? It is known that SLN does
not correspond to HvSPY (Robertson et al., 1998), but there is no
evidence yet concerning its relationship to GAI or RGA. Scott (1990)
suggested that (semi)-dominant GA "insensitive" mutants (encoded by
gai, Rht3, and D8) might involve the
same gene that is affected in recessive constitutive GA-response
mutants such as sln1: GAI would involve a gain of function
(a negative regulator whose activity was no longer regulated by GA),
whereas the mutant SLN would involve a loss of function. The cloning of
GAI and RGA should allow their relationship to SLN to be investigated.
| |
FOOTNOTES |
*
Corresponding author; e-mail peter.chandler{at}pi.csiro.au;
fax 61-2-6246-5000.
Received December 1, 1998;
accepted February 21, 1999.
| |
ABBREVIATIONS |
Abbreviations:
L1, first leaf or leaf one.
LERmax, maximal leaf-elongation rate.
| |
ACKNOWLEDGMENTS |
We thank Bruce Twitchin in the laboratory of Prof. L.N. Mander
for the generous supply of pure GA1 and
GA3, Mark Cmiel for skilled technical assistance,
Dr. Jonathan Weyers for helpful discussions, and R. King, F. Gubler, N. Paterson, and J. Weyers for comments on the manuscript.
| |
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Abstract
Introduction